RT Journal Article SR Electronic T1 The Use of 3-[2-(N,N-Diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) as a Specific CYP2D6 Probe in Human Liver Microsomes JF Drug Metabolism and Disposition JO Drug Metab Dispos FD American Society for Pharmacology and Experimental Therapeutics SP 1196 OP 1200 VO 29 IS 9 A1 Nathalie Chauret A1 Barry Dobbs A1 Rebecca L. Lackman A1 Kevin Bateman A1 Deborah A. Nicoll-Griffith A1 David M. Stresser A1 Joseph M. Ackermann A1 Stephanie D. Turner A1 Vaughn P. Miller A1 Charles L. Crespi YR 2001 UL http://dmd.aspetjournals.org/content/29/9/1196.abstract AB Recently, a novel nonfluorescent probe 3-[2-(N,N-diethyl-N-methylammonium)-ethyl]-7-methoxy-4-methylcoumarin (AMMC), which produces a fluorescent metabolite AMHC (3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-hydroxy-4-methylcoumarin) was used with microsomes containing recombinant enzymes (rCYP) to monitor CYP2D6 inhibition in a microtiter plate assay. This article describes the studies that were performed in human liver microsomes (HLM) to establish the selectivity of AMMC toward CYP2D6. Metabolism studies in HLM showed that AMMC was converted to one metabolite identified by mass spectrometry as AMHC. Kinetic studies indicated an apparent Km of 3 μM with aVmax of 20 pmol/min · mg of protein for the O-demethylation reaction. TheO-demethylation of AMMC in HLM was inhibited significantly in the presence of a CYP2D6 inhibitory antibody. Using a panel of various HLM preparations (n = 12), a good correlation (r2 = 0.95) was obtained between AMMC O-demethylation and bufuralol metabolism, a known CYP2D6 substrate, but not with probes for the other major xenobiotic metabolizing CYPs. Finally, only rCYP2D6 showed detectable metabolism in experiments conducted with rCYPs using AMMC at a concentration of 1.5 μM (near Km). However, at a concentration of 25 μM AMMC, rCYP1A also contributed significantly to the formation of AMHC. Knowing the experimental conditions under which AMMC was selective for CYP2D6, a microtiter assay was developed to study the inhibition of various compounds in HLM using the fluorescence of AMHC as an indication of CYP2D6 activity. The inhibition potential of various chemicals was found to be comparable to those determined using the standard CYP2D6 probe, bufuralol, which requires high-performance liquid chromatography separation for the analysis of its CYP2D6-mediated 1′-hydoxylated metabolite. The American Society for Pharmacology and Experimental Therapeutics